Biomedical subjects
Edward Harris
Publications and source records attributed to Edward Harris.
RabD, a Dictyostelium Rab14-related GTPase, regulates phagocytosis and homotypic phagosome and lysosome fusion.
RabD, a Dictyostelium Rab14-related GTPase, localizes in the endo-lysosomal pathway and contractile vacuole system of membranes. Cell lines expressing dominant-negative RabD were defective in endocytosis, endosomal membrane flow and homotypic lysosome fusion. In support of a role for RabD in fusion, cells overexpressing constitutively active RabD(Q67L) accumulated enlarged hydrolase-rich acidic vesicles ringed with GFP-RabD, consistent with RabD directly regulating lysosome fusion. To determine whether RabD also regulated phagocytosis and/or homotypic phagosome fusion (a process stimulated by many intracellular pathogens), cells overexpressing dominant-active (RabD(Q67L)) or dominant-negative (Rab(N121I)) RabD were analyzed microscopically and biochemically. The rate of phagocytosis was increased two-fold in RabD(Q67L)-expressing cells and reduced by 50% in RabD(N121I)-expressing cells compared with control cells. To examine the role of RabD in the formation of multiparticle phagosomes, we performed a series of pulse-chase experiments using fluorescently labeled bacteria and fluorescent latex beads. The rate of fusion of newly formed phagosomes was five times higher in the RabD(Q67L)-expressing cells and reduced by over 50% in RabD(N121I)-expressing cells as compared with control cells. GFP-RabD(Q67L) was found to ring multiparticle spacious phagosomes, which supports a direct role for this protein in regulating fusion. Inhibition of PI 3-kinase activity, which is known to regulate phagosome fusion in the wild-type cells, reduced the rate of phagosome fusion in RabD(Q67L+) cells, indicating that RabD acted upstream of or parallel with PI 3-kinase. We hypothesize that RabD and, possibly, Rab14, a related GTPase that associates with phagosomes in mammalian cells, are important regulators of homotypic phagosome and endo-lysosome fusion.
Dictyostelium LvsB mutants model the lysosomal defects associated with Chediak-Higashi syndrome.
Chediak-Higashi syndrome is a genetic disorder caused by mutations in a gene encoding a protein named LYST in humans ("lysosomal trafficking regulator") or Beige in mice. A prominent feature of this disease is the accumulation of enlarged lysosome-related granules in a variety of cells. The genome of Dictyostelium discoideum contains six genes encoding proteins that are related to LYST/Beige in amino acid sequence, and disruption of one of these genes, lvsA (large volume sphere), results in profound defects in cytokinesis. To better understand the function of this family of proteins in membrane trafficking, we have analyzed mutants disrupted in lvsA, lvsB, lvsC, lvsD, lvsE, and lvsF. Of all these, only lvsA and lvsB mutants displayed interesting phenotypes in our assays. lvsA-null cells exhibited defects in phagocytosis and contained abnormal looking contractile vacuole membranes. Loss of LvsB, the Dictyostelium protein most similar to LYST/Beige, resulted in the formation of enlarged vesicles that by multiple criteria appeared to be acidic lysosomes. The rates of endocytosis, phagocytosis, and fluid phase exocytosis were normal in lvsB-null cells. Also, the rates of processing and the efficiency of targeting of lysosomal alpha-mannosidase were normal, although lvsB mutants inefficiently retained alpha-mannosidase, as well as two other lysosomal cysteine proteinases. Finally, results of pulse-chase experiments indicated that an increase in fusion rates accounted for the enlarged lysosomes in lvsB-null cells, suggesting that LvsB acts as a negative regulator of fusion. Our results support the notion that LvsB/LYST/Beige function in a similar manner to regulate lysosome biogenesis.